Non-coding RNA forms the cell?s machineries for protein synthesis and splicing, and acts during all stages of gene expression. All RNAs must begin to fold and interact with their protein partners as they are being synthesized. Improper expression, assembly and localization of non-coding RNA has been linked to cancer, neurodegeneration and developmental abnormalities. Although thousands of new non-coding RNAs have been discovered, we know little about how they physically carry out their functions. The goal of this research program is to define principles of RNA-protein assembly and dynamics that can predict how large RNA-protein complexes are formed and how small non-coding RNAs rapidly search out their regulatory targets. These concepts will be addressed using two model systems: ribosome biosynthesis and post- transcriptional regulation by bacterial small RNA and Hfq. Ribosomes are an important example of co-transcriptional assembly because ribosomes are continually synthesized in growing cells and tightly regulated by growth rate and by stress response pathways. New single molecule fluorescence and in-cell RNA structure probing approaches will be used to visualize how RNA folding and protein binding is coupled to transcription. Small non-coding RNAs base pair with mRNA to control mRNA expression, and are a ubiquitous form of RNA-based regulation. Single molecule fluorescence, native mass spectrometry and bacterial reporter assays will reveal how the Hfq RNA chaperone recycles small non-coding RNA in bacteria, and increases the rate at which these RNAs find their proper targets. The results of this research will lead to new concepts related to how RNA-protein complexes assemble faithfully during normal growth and why certain RNAs are vulnerable to mutation, stress, or competition from foreign RNA.